The Molecular Mechanism Research Of MIR319under Cold Stress In Rice

Cold stress, adversely affecting the plant growth and development, can significantly constraint the spatial distribution and agricultural productivity. Cultivating new psychrotolerant varieties is the most effective way to develop agricultural yield of rice. Since cold stress response is a process that requires the participation of many genes, the effect of ectopic expression for a single functional protein to increase cold tolerance is very limited. Thus, it’s difficult to meet the actual requirements of high agricultural production. MicroRNAs（miRNAs） are approximate21nucleotide（nt） single-stranded RNAs that play important roles in plants biological processes by targeting mRNAs cleavage or translational repression. miR319was reported to be involved in the regulation of plant growth and development. In our previous study, we found miR319was down-regulated under cold stress by microarray.In this study, we firstly confined miR319detected in the microarrays as cold responsive miRNAs with semi-RT PCR and real-time PCR. Then, over-expression of miR319was done with the Agrobacterium co-cultivation method to check their effect on cold response. After identifying miRNA-mRNA pairs by combining bioinformatics tools （psRNAtarget） and microarray data analysis, we detected the expression of putative target gene in over-expression lines. Lastly the expression of cold-stress-responstive gene in miR319b over-expression lines was analyzed to identify the molecular regulatory mechanisms of miR319b in response to cold stress. The main results in this study are as follows:1. Verification of miRNA in response to cold stress in riceWe used RT-PCR to analysis the expression of pre-miR319under cold stress and found miR319a and miR319b were up-regulated at0.5h and then significantly down-regulated within24h.2. Functional analysis of miR319by over-expression in riceWe constructed pre-miR319a/b plant over-expression vectors and accomplished the Agrobacterium mediated genetic transformation of rice callus. Through PCR, Southern blotting and RT-PCR detection analysis, we obtained more than three transgenic lines for each gene. Then, we used stem-loop quantitative real-time PCR to detect the mature miRNA expression levels. The level of miR319b was increased in pre-miR319b transgenic lines, but level of miR319a had no change comparing with the wild-type. There was no obvious change on cold-tolerance between pre-miR319a transgenic rice and wild-type rice, while miR319b over-expression lines showed shorter internode, improved cold tolerance.3. Prediction and biological verification of miR319targetsUsing target prediction and relationship check, we got26target genes. Then, quantitative real-time PCR was used to analysis the expression levels in over-expression lines. We verified two target genes, Os₀3g57190（FCF6） and Os₀7g05720（OsTCP21）, both of which were TCP transcriptional factors involved in cell proliferation and differentiation.4. The molecular mechanism of MiR319b in cold stress responseWe analyzed the cold-related gene expression levels in the miR319b over-expression plants and wild-type. DREB/CBF transcription factor genes and TPPs were up-regulated, OsCPTl was down-regulated. TPPs were reported to be cold stress-induced, and over-expression of the TPPs could accumulate trehalose, an important kinds of osmotic protective materials, and improve the cold resistance. The CBF/DREB transcription factor could activate the expression of many COR genes. They can bind to the cold-and dehydration-responsive DNA regulatory elements（DRE）, also termed C-repeats（CRTs）, which is sufficient to induce transcription under cold stress. OsCPT1was induced by cold stress in OsMYB3R-2gene transgenic lines, which improved transgenic plants resistance to cold. However, in this study, OsCPTl showed opposite expression in the miR319b over-expression plants, and the molecular mechanisms regulation of miR319b under cold tolerance was complex, which need to be further researched.